Bottom Line:
The quantification of allergens in food including baked food matrices is of great interest.The peptide VLPVPQK was selected as the signature peptide for bovine β-casein because of the high sensitivity.A stable isotope-labelled internal standard was designed to adjust the instability of sample pre-treatment and ionisation caused by matrix effect.

ABSTRACTThe quantification of allergens in food including baked food matrices is of great interest. The aim of the present study was to describe a non-immunologic method to quantify bovine β-casein using ultra-performance liquid chromatography tandem triple quadrupole mass spectrometry (UPLC-TQ-MS/MS) in multiple reaction monitoring (MRM) mode. Eight of 10 theoretical peptides from β-casein after tryptic digestion were compared and MRM methods were developed to determine five signature peptides. The peptide VLPVPQK was selected as the signature peptide for bovine β-casein because of the high sensitivity. A stable isotope-labelled internal standard was designed to adjust the instability of sample pre-treatment and ionisation caused by matrix effect. Using the present suspension digestion method, the native and denatured β-casein could be digested to release the signature peptide at the maximum extent. The UPLC-TQ-MS/MS method developed based on a tryptic signature peptide led to a reliable determination of bovine β-casein allergen in baked food matrices at a low quantitation level down to 500 μg kg(-1) with a satisfactory accuracy (< 8.9%) and recovery (98.8% ± 2.6% to 106.7% ± 3.0%).

Figure 0003: Relative peak areas normalised to a signature peptide standard and IS1, IS2 in different food matrices (n = 3). A total of 0.1 g negative sample and commonly used baking ingredients (wheat flour, sugar, salt, peanut oil, hen’s eggs, cacao and coconut) were weighed into the Eppendorf caps and mixed with 100 µl of 10 µg ml–1 bovine β-casein. After spiking of 10 µl IS1 (0.25 µg ml–1) and IS2 (1 µg ml–1) into the caps, the mixtures were digested as described in the ‘Tryptic digestion and peptide extraction’ section. The solutions of bovine β-casein and ISs at the same concentration were digested as a standard, and its peak area was set as 100% abundance.

Mentions:
The results (Figure 3) showed that the relative peak areas of signature peptide (VLPVPQK) in the presence of different food matrix were similar to the results of the first experiment (Figure 2) except in egg, cacao and peanut oil. There was almost no signature peptide detected in the presence of egg because egg contained ovomucoid, which was known for its trypsin inhibitor property. Cacao is rich in polyphenols, which can interact with proteins to form complex in solution. It was speculated that polyphenols in cacao might inhibit the digestion process because of their interaction with the bovine β-casein and the trypsin. Figure 3 also showed that in the presence of peanut oil, the content of signature peptide was about 40% of the signature peptide in the standard mix. This could not be simply explained by inhibition of the tryptic digestion because the IS2 was not affected by the presence of peanut oil in the same tryptic digestion. Only signature peptide was affected by the peanut oil, possibly because the non-identical behaviour between protein and IS peptide in an oily sample. The digestion steps releasing the corresponding signature peptides from target proteins require the cleavage sites to be solvent accessible for proteolysis to occur. Nevertheless, the high oil contents in food matrices affect the solvent accessible levels of target proteins and IS in different extent. The usage of stable isotope-labelled protein might solve this problem (Pritchard et al. 2014). However, it is worth noting that the negative sample in this study contained about 16% peanut oil. The relative peak area of signature peptide in negative sample in experiment 2 was almost identical to the results of experiment 1. All these results showed that the signature peptide of β-casein (VLPVPQK) could work well with the negative sample and the wheat flour, sugar, salt and coconut. This signature peptide might not work well with baked foodstuff containing egg, cacao or high content of oil.Figure 3.

Figure 0003: Relative peak areas normalised to a signature peptide standard and IS1, IS2 in different food matrices (n = 3). A total of 0.1 g negative sample and commonly used baking ingredients (wheat flour, sugar, salt, peanut oil, hen’s eggs, cacao and coconut) were weighed into the Eppendorf caps and mixed with 100 µl of 10 µg ml–1 bovine β-casein. After spiking of 10 µl IS1 (0.25 µg ml–1) and IS2 (1 µg ml–1) into the caps, the mixtures were digested as described in the ‘Tryptic digestion and peptide extraction’ section. The solutions of bovine β-casein and ISs at the same concentration were digested as a standard, and its peak area was set as 100% abundance.

Mentions:
The results (Figure 3) showed that the relative peak areas of signature peptide (VLPVPQK) in the presence of different food matrix were similar to the results of the first experiment (Figure 2) except in egg, cacao and peanut oil. There was almost no signature peptide detected in the presence of egg because egg contained ovomucoid, which was known for its trypsin inhibitor property. Cacao is rich in polyphenols, which can interact with proteins to form complex in solution. It was speculated that polyphenols in cacao might inhibit the digestion process because of their interaction with the bovine β-casein and the trypsin. Figure 3 also showed that in the presence of peanut oil, the content of signature peptide was about 40% of the signature peptide in the standard mix. This could not be simply explained by inhibition of the tryptic digestion because the IS2 was not affected by the presence of peanut oil in the same tryptic digestion. Only signature peptide was affected by the peanut oil, possibly because the non-identical behaviour between protein and IS peptide in an oily sample. The digestion steps releasing the corresponding signature peptides from target proteins require the cleavage sites to be solvent accessible for proteolysis to occur. Nevertheless, the high oil contents in food matrices affect the solvent accessible levels of target proteins and IS in different extent. The usage of stable isotope-labelled protein might solve this problem (Pritchard et al. 2014). However, it is worth noting that the negative sample in this study contained about 16% peanut oil. The relative peak area of signature peptide in negative sample in experiment 2 was almost identical to the results of experiment 1. All these results showed that the signature peptide of β-casein (VLPVPQK) could work well with the negative sample and the wheat flour, sugar, salt and coconut. This signature peptide might not work well with baked foodstuff containing egg, cacao or high content of oil.Figure 3.

Bottom Line:
The quantification of allergens in food including baked food matrices is of great interest.The peptide VLPVPQK was selected as the signature peptide for bovine β-casein because of the high sensitivity.A stable isotope-labelled internal standard was designed to adjust the instability of sample pre-treatment and ionisation caused by matrix effect.

ABSTRACTThe quantification of allergens in food including baked food matrices is of great interest. The aim of the present study was to describe a non-immunologic method to quantify bovine β-casein using ultra-performance liquid chromatography tandem triple quadrupole mass spectrometry (UPLC-TQ-MS/MS) in multiple reaction monitoring (MRM) mode. Eight of 10 theoretical peptides from β-casein after tryptic digestion were compared and MRM methods were developed to determine five signature peptides. The peptide VLPVPQK was selected as the signature peptide for bovine β-casein because of the high sensitivity. A stable isotope-labelled internal standard was designed to adjust the instability of sample pre-treatment and ionisation caused by matrix effect. Using the present suspension digestion method, the native and denatured β-casein could be digested to release the signature peptide at the maximum extent. The UPLC-TQ-MS/MS method developed based on a tryptic signature peptide led to a reliable determination of bovine β-casein allergen in baked food matrices at a low quantitation level down to 500 μg kg(-1) with a satisfactory accuracy (< 8.9%) and recovery (98.8% ± 2.6% to 106.7% ± 3.0%).